Methylated tumor suppressor gene promoters that are commonly observed in clinically-apparent lung malignancies have also been detected in non-malignant and pre-malignant lung epithelia in asymptomatic tobacco smokers. These methylation markers of early carcinogenesis, if prospectively predictive of the emergence of clinical disease, could be applied as biomarkers of cancer risk. The laboratory has developed and published a simplified method of generating promoter methylation maps at single-base resolution across kilobase stretches of gene promoter DNA, without the need for cloning, by a tag-adaptation of bisulfite genomic sequencing (tBGS). Our pilot studies have reliably identified DNA in exhaled breath condensate (EBC). The coupling of tBGS methylation maps to EBC samples has yielded the first examples of DNA methylation assays from exhaled breath. The laboratory is now able to multiplex this assay for a panel of six gene promoters. This R21 proposal is designed to test the potential of that exhaled DNA to be informative for: (i) airway site of origin;(ii) tobacco smoke exposure;and (iii) lung cancer case-control status. In order to determine the origin of exhaled breath DNA, those levels of the airway that may plausibly contribute to exhaled breath DNA methylation patterns (buccal, bronchial, alveolar) will be directly compared to EBC from the same 75 patients, for methylation patterns for six cancer-relevant tumor suppressor genes at each of these levels. Multivariate comparisons to tobacco smoke exposure, to lung cancer case-control status, and to tumor characteristics, will also be made in 150 subjects, to pilot test the potential clinical utility of this non-invasive biomarker. PUBLIC HEALTH RELEVANCE: The project will pilot test the validity of exhaled breath DNA methylation as a new class of non-invasive lung carcinogenesis biomarker. If validated, this test would enable the identification of individuals at particularly high risk for lung cancer, for the purpose of focusing prevention and early detection efforts on those most likely to benefit.